Extracellular phospholipases facilitate invasion of, and escape from, host cells during the infection caused by a number of microbial pathogens, ranging from bacteria to protozoa. We have found evidence that phospholipase is a virulence factor in C. albicans. Blood isolates from patients secrete significantly more phospholipases than do commensal isolates. When we infected mice intravenously with these blood isolates, the survival of the mice was inversely related to the phospholipase production. Also, we studied two other strains of C. albicans that differed markedly in their ability to cause disseminated infection in the newborn mouse model. The invasive strain produced over 3-fold more phospholipase than did the non-invasive strain, again indicating that the secretion of phospholipase is important in candidal invasion. We determined that the invasive strain secreted a phospholipase B and/or a lysophospholipase suggesting that one or both of these enzymes is involved in the virulence of this organism. We will now determine if the observed relationships between increased phospholipase production and increased candidal pathogenicity indicate cause and effect by constructing isogenic strains that differ in only one gene product. Towards this end, we have cloned a gene from C. albicans that encodes a putative extracellular phospholipase and we have expressed this gene in Saccharomyces cerevisiae. This technique allows for the isolation of independent virulence factors, which is necessary for the critical evaluation of any putative candidal virulence factor. Additionally, we have initiated a biochemical approach to complement the molecular genetic approach. We have isolated and partially purified a native phospholipase B from C. albicans. The purpose of these experiments is to definitively delineate the role of extracellular phospholipases in the pathogenesis of candidal infections by: 1. characterizing the phospholipases secreted by C. albicans, 2. cloning the genes responsible for the candidal phospholipase/s that is/are involved-in virulence, 3. using targeted mutagenesis to produce well-defined null mutants of C. albicans that have the specific phospholipase gene deleted, and 4. comparing these isogenic strains, which are deficient in phospholipase production, with the parent strain for their capacity to establish and maintain hematogenously disseminated infections in vivo, adhere to and damage endothelial cells, and resist neutrophil killing in vitro. Once the role of the phospholipases and the genes responsible for their production in the virulence of this emerging nosocomial pathogen have been defined, appropriate therapeutic strategies can be developed. This strategy will define the role of phospholipase in virulence as well as provide a powerful method to determine the contribution of other individual virulence factors in a situation where multiple factors are likely required for virulence.
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